Humans grow corn for food and energy applications. Insects eat and damage corn plants and thereby undermine these human efforts.
Current in-plant transgenic control of these pests is achieved through plant expression of a crystal (Cry) delta endotoxin gene coding for the Cry1Fa protein from Bacillus thuringiensis. Cry1Fa is the protein toxin currently in the Herculex™ brand of Dow AgroSciences transgenic corn seeds (Herculex, Herculex-Extra, and Herculex-RW) that are resistant to fall armyworm (FAW, Spodoptera frugiperda) and European corn borer (ECB, Ostrinia nubilalis) insect pests. This protein works by binding to specific receptor(s) located in the midgut of insects, and forms pores within the gut cells. The formation of these pores prevents insects from regulating osmotic balance which results in their death.
However, some are concerned that insects might be able to develop resistance to the action of Cry1Fa through genetic alterations of the receptors within their gut that bind Cry1Fa. Insects that produce receptors with a reduced ability to bind Cry1Fa can be resistant to the activity of Cry1Fa, and thus survive on plants that express this protein.
With a single Cry toxin continuously present in the plant during growth conditions, there is concern that insects could develop resistance to the activity of this protein through genetic alterations of the receptor that binds Cry1Fa toxin in the insect gut. Reductions in toxin binding due to these alterations in the receptor would lead to reduced toxicity of the Cry1Fa possibly leading to eventual decreased effectiveness of the protein when expressed in a crop. See e.g. US 2009 0313717, which relates to a Cry2 protein plus a Vip3Aa, Cry1F, or Cry1A for control of Helicoverpa zea or armigera. WO 2009 132850 relates to Cry1F or Cry1A and Vip3Aa for controlling Spodoptera frugiperda. US 2008 0311096 relates to Cry1Ab for controlling Cry1F-resistant ECB.
Additional Cry toxins are listed at the website of the official B.t. nomenclature committee (Crickmore et al.; lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/). See Appendix A, attached. There are currently nearly 60 main groups of “Cry” toxins (Cry1-Cry59), with additional Cyt toxins and VIP toxins and the like. Many of each numeric group have capital-letter subgroups, and the capital letter subgroups have lower-cased letter sub-subgroups. (Cry1 has A-L, and Cry1A has a-i, for example).
The van Frankenhuyzen (2009) reference (J. Invert. Pathol. 101:1-16), for example, illustrates that there are many target pests and a great number of toxins that could potentially be selected to control the target pests. See e.g. FIG. 3 of van Frankenhuyzen. One (among many) pests that could be targeted would include Ostrinia nubilalis, and for this insect, FIG. 3 of van Frankenhuyzen shows 17 toxins that are active against ECB, and one that is possibly active. This is not an exhaustive list of the options.
FIG. 3 of van Frankenhuyzen also illustrates that each Cry protein has a unique spectrum of activity—they are active against some insects but not others. Cry proteins typically bind receptors on cells in the insect gut, and this is one factor that influences the spectrum of activity. Receptors for one Cry protein can be found in some insects but not in others; a given insect might have receptors for one or more Cry proteins but not for other Cry proteins.
Given many possible insects to target, and many possible Cry proteins that could be active against any given insect, numbers alone illustrate the complexity of the problem of resistance management. Considering just the 18 proteins identified by van Frankenhuyzen as active or possibly against ECB, this would allow for hundreds of possible pairs of toxins to test in combination.
In addition, assaying for competitive/non-competitive binding is no easy task. It can involve radio-active labeling and assaying for displacement of radioactively labeled proteins. This in and of itself can be a complex art.
Attempting to use resistant insects, directly, is also complicated. Resistant strains of insects would have to be developed against a given protein. Siqueira (June 2004; J. Econ. Entomol., 97(3):1049-1057) states (in the abstract) that “ . . . tests for cross-resistance among different toxins have been limited by a lack of resistant colonies.” This illustrates difficulties with obtaining resistant insect strains for assaying proteins for resistance management potential. When pairs of proteins are involved, either protein could be used in an attempt to screen for the development of resistant insects.
Siqueira also states, in the abstract, that selection with Cry1Ab (i.e., developing colonies of ECB that are resistant to Cry1Ab) “ . . . resulted in decreased susceptibility to a number of other toxins . . . . ” This illustrates the phenomenon of cross-resistance. Cry1Ab-resistant ECB were cross-resistant to “a number of other toxins.”
Thus, selecting two proteins that are active against the same (non-resistant) insect is a mere starting point of the analysis, if resistance issues are to be addressed. Activity levels against non-resistant insects is another factor. FIG. 11 of van Frankenhuyzen shows that even among a group of 12 toxins selected for testing against ECB (non-resistant), other Cry proteins (such as Cry1Ac, Cry1Bb, and Cry2Aa) could be more active than the ones now claimed for controlling ECB.